Environmental Biotechnology - Jordening and Winter

References 201

tance in the production of gases which have an inhibitory effect on methane generation like H2S and NH3.

The influence of hydrostatic pressure on biogas production (Section 6.6) should be taken into consideration for most large technical scale bioreactors with a height of more than 5 m.

List of Symbols

Greek

Special Symbols of Chemical Components

204 7 Aerobic Degradation of Recalcitrant Organic Compounds by Microorganisms

Fig. 7.1 Main characteristics of aerobic degradation of hydrocarbons: processes associated with growth of microorganisms.

carbon and energy. This process results in a complete degradation (mineralization) of organic pollutants, as demonstrated in Section 7.2.2. Cometabolism is defined as the metabolism of an organic compound in the presence of a growth substrate that is used as the primary carbon and energy source. The principle is explained in Section 7.2.4.

Key enzymatic reactions of aerobic biodegradation are oxidations catalyzed by oxygenases and peroxidases. Oxygenases are oxidoreductases that use O2 to incorporate oxygen into the substrate. Degradative organisms need oxygen at two metabolic sites

– the initial attack on the substrate and the end of the respiratory chain (Fig. 7.1). Certain higher fungi have developed a unique oxidative system for the degradation of lignin based on extracellular ligninolytic peroxidases and laccases. This enzymatic system possesses increasing significance for the cometabolic degradation of persistent organopollutants. Thus, the basidiomycetous fungi require deeper insights and extensive consideration. Therefore, this chapter is divided into two sections: bacterial and fungal degradation.

7.2 Principles of Bacterial Degradation 205

7.2

Principles of Bacterial Degradation

7.2.1

Typical Aerobic Degrading Bacteria

The predominant degraders of organopollutants in the oxic zone of contaminated areas are chemo-organotrophic species, which are able to use a huge number of natural and xenobiotic compounds as carbon sources and as electron donors for the generation of energy. Although many bacteria are able to metabolize organic pollutants, a single bacterial species does not possess the enzymatic capability to degrade all or even most of the organic compounds in a polluted soil. Mixed microbial communities have the most powerful biodegradative potential, because the genetic information of more than one organism is necessary to degrade the complex mixtures of organic compounds present in contaminated areas. The genetic potential and certain environmental factors such as temperature, pH, and available nitrogen and phosphorous sources, therefore, seem to determine the rate and the extend of degradation.

The predominant bacteria in polluted soils belong to a spectrum of genera and species (Table 7.1). The table lists only bacteria that can be cultured on nutrient-rich media. We have to consider that the majority of bacteria present in soils cannot yet be cultivated in the laboratory.

The pseudomonads, aerobic gram-negative rods that never show fermentative activity, seem to have the highest degradative potential, e.g., Pseudomonas putida and P. fluorescens. Further important degraders of organic pollutants are found within the genera Comamonas, Burkholderia, and Xanthomonas. Some species utilize >100 different organic compounds as carbon sources. The immense potential of the pseudomonads does not solely depend on the catabolic enzymes, but also on their capability for metabolic regulation (Houghton and Shanley 1994). A second important group of degrading bacteria are the gram-positive rhodococci and coryneform bacteria. Many species, now classified as Rhodococcus spp., were originally described as

Nocardia spp., Mycobacterium spp., and Corynebacterium spp.. Rhodococci are aero-

Table 7.1 Predominant bacteria in soil samples polluted with aliphatic and aromatic hydrocarbons, polycyclic aromatic hydrocarbons and chlorinated compounds.a

aBacteria have been reclassified based on phylogenetic markers, resulting in changes in some genera and species; thus, the names of species are not mentioned.

7.2 Principles of Bacterial Degradation 207

Pathways of alkane degradation. The main pathway is their terminal oxidation to fatty acids catalyzed by n-alkane monooxygenase, alcohol dehydrogenase, and ) aldehyde dehydrogenase.

orate rapidly from petroleum-contaminated sites. Oxidation of alkanes is classified as terminal or diterminal. Monoterminal oxidation is the main pathway and proceeds by formation of the corresponding alcohol, aldehyde, and fatty acid. Beta oxidation of fatty acids results in the formation of acetyl-CoA. n-Alkanes having an odd number of carbon atoms are degraded to propionyl-CoA, which is in turn carboxylated to methylmalonyl-CoA and further converted to succinyl-CoA. Fatty acids having a physiological chain length may be directly incorporated into membrane lipids, but most degradation products are fed into the tricarboxylic acid cycle. Subterminal oxidation occurs with lower (C3–C6) and longer alkanes, with formation of a secondary alcohol and subsequently of a ketone. Unsaturated 1-alkenes are oxidized at the saturated end of the chains. A minor pathway proceeds via an epoxide, which is converted to a fatty acid. Branching generally reduces the rate of biodegradation. Methyl side groups do not noticeably decrease the biodegradability, whereas complex

208 7 Aerobic Degradation of Recalcitrant Organic Compounds by Microorganisms

branched chains, e.g., the tertiary butyl group, hinder the action of the degradative enzymes.

Cyclic alkanes represent minor components of mineral oil and are relatively resistant to microbial attack. The absence of an exposed terminal methyl group complicates the primary attack. A few species can use cyclohexane as a sole carbon source, but it is more commonly cometabolized by mixed cultures. The pathway of cyclohexane degradation is shown in Figure 7.4. In general, the presence of alkyl sidechains on cycloalkanes facilitates their degradation.

Aliphatic hydrocarbons become less water soluble with increasing chain length; those with a chain length of C12 or more are virtually water-insoluble. Two mechanisms are involved in microbial uptake of these lipophilic substrates: attachment of microbial cells to oil droplets and production of biosurfactants. The uptake mechanism linked to attachment of the cells is still unknown, but the effect of biosurfac-

Fig. 7.4 Metabolic pathways for degradation of cycloaliphatic compounds (cycloparaffins).

2107 Aerobic Degradation of Recalcitrant Organic Compounds by Microorganisms

7.2.3

Diversity of Aromatic Compounds: Unity of Catabolic Processes

Aromatic hydrocarbons, e.g., benzene, toluene, ethylbenzene and xylenes (BTEX compounds), and naphthalene, belong to the large-volume petrochemicals, which are widely used as fuels and industrial solvents. Phenols and chlorophenols are released into the environment as products and waste materials from industry. Aromatic compounds are formed in large amounts by all organisms, e.g., as aromatic amino acids, phenols, or hydroquinones/quinones. Thus, it is not surprising that many microorganisms have evolved catabolic pathways to degrade aromatic compounds. In general, man-made organic chemicals (xenobiotics) can be degraded by microorganisms when they are similar to natural compounds. The range of man-made aromatics shown in Figure 7.6 can be converted enzymatically to the natural intermediates of degradation: catechol and protecatechuate. In general, benzene and related compounds are characterized by greater thermodynamic stability than aliphatics are. The first step in benzene oxidation is a hydroxylation that is catalyzed by a dioxygenase (Fig. 7.2). The product, a diol, is then converted to catechol by a dehydrogenase. These initial reactions, hydroxylation and dehydrogenation, are also common

Degradation of a broad spectrum of natural and xenobiotic aromatic compounds into two central intermediates: catechol and protocatechuate.